Abstract Skp2 is the substrate specificity factor of the SCFSkp2 E3 ligase involved in cell cycle progression through degradation of its ubiquitin targets such as p21, p27 and FOXO1. Since most of these substrates are tumor suppressor proteins, Skp2 functions as an oncogene and Skp2 overexpression is frequently observed in prostate carcinomas. However, the exact molecular mechanisms by which Skp2 induces prostate tumor growth and whether targeting Skp2 could be used as an efficient anti-prostate cancer therapy have not been fully elucidated. We recently reported that human Skp2 abundance and oncogenic functions are regulated by p300- mediated acetylation that could be antagonized by SIRT3. Moreover, expression of an acetylation-mimetic mutant of Skp2 promotes in vivo tumorigenesis in a xenograft model. We also obtained preliminary results showing that in multiple prostate cancer cell lines, Skp2 acetylation could be induced by androgen, and constitutive Skp2 acetylation might contribute to castration resistance while the underlying mechanism remains unclear. Furthermore, we identified IDH1 as a putative substrate of SCFSkp2, therefore revealing a novel role of Skp2 in cancer cell metabolism control that might underscore its oncogenic functions. Based on our preliminary data, we hypothesize that Skp2 oncogenic function is governed by acetylation, and aberrantly elevated Skp2 acetylation promotes prostate tumorigenesis and castration resistance in part by targeting downstream substrates such as IDH1 for degradation. Here, we intend to test our hypotheses by accomplishing three specific aims. In Aim #1, we will determine the molecular mechanisms by which the androgen/AR signaling pathway governs Skp2 oncogenic activity in the prostate cancer setting by regulating Skp2 acetylation. These studies will provide a novel mechanism on how androgen-induced acetylation of Skp2 regulates its oncogenic functions in the prostate cancer setting. It will also shed important insights into whether Skp2 acetylation could lead to the development of castration resistance. In Aim #2, we will examine whether acetylation of Skp2 could affect in vivo prostate cancer development using orthotopic and various engineered mouse models. These in vivo studies will significantly expand our knowledge of the regulation of Skp2 oncogenic functions by the p300/SIRT3 regulatory circuit. More importantly, we will carry out preclinical trials with MLN4924 and Skpin or compound #25 to examine if inhibiting Skp2 activity could efficiently retard in vivo prostate tumorigenesis and restore castration sensitivity. In Aim #3, we will determine the novel molecular mechanisms through which IDH1 stability is modulated by Skp2 in a Cdk2 and cell cycle dependent manner. Moreover, we will investigate whether inhibiting the Cdk2/Skp2 signaling axis could suppress prostate tumorigenesis in part by stabilizing IDH1. We believe that our proposed studies will not only provide a better molecular understanding of how Skp2 oncogenic activity is governed in vivo, but also provide direct evidence to evaluate if inactivating Skp2 could efficiently suppress prostate cancer development, leading to better treatment of this deadly disease.